Persistent immune activation in HIV-1 infected individuals, characterized by increased levels of pro- inflammatory cytokines, such as IL-7, IL-6, IFN-?, TNF-??, and expression of activation markers, including CD69, is associated with a lack of T cell regeneration despite successful antiretroviral therapy. Lack of T cell regeneration is likely due in part to a decrease in entry of hematopoietic stem cells into the thymus and egress of naive T cells from the thymus to the periphery. However, there are no data available elucidating these processes in humans. From studies in the mouse it has become apparent that Sphingosine-1-phosphate (S1P) and one of its receptors, S1P receptor-1 (S1P-R1) play essential roles in egress of naive T cells from the thymus to the periphery as well as egress of memory T cells from the secondary lymphoid tissues. There is also evidence that S1P controls expression of chemokines like CCL25 by thymic endothelial and cortical epithelial cells and thereby entry of precursors into the murine thymus. In addition, S1P enhances SDF-1??/CXCR4 migration of human CD34+ cells. Together these data suggest that S1P plays an important role in entry of CD34+ precursors into the thymus as well as egress of naive T cells from the thymus to the periphery. In addition to S1P receptor expression, the CD69 molecule has been found to play a role in regulating thymocyte egress. CD69 is known as an early activation molecule in the periphery. In the thymus CD69 is expressed during positive and negative selection, but is not present on recent thymic emigrants. Our novel preliminary data in the human thymus show that only mature CD69- thymocytes (CD3hiCD27+CD45RA+ CD62L+CD69-), which are phenotypically identical to naive peripheral blood T cells, can respond to S1P and emigrate out of the thymus in response to S1P. This effect is inhibited by FTY720, an S1P-R agonist. Interestingly, our preliminary data also shows that CD69 is upregulated on CD34+ progenitor cells after exposure to IL-7 and IFN-??. Thus, based on murine data in the literature and our preliminary data with human CD34+ cells and thymocytes we hypothesize that HIV-1 induced immune activation increases expression of CD69 leading to downregulation of S1P-R1, thereby interfering with entry of CD34+ cells into and egress of mature CD69- thymocytes out of the thymus to the periphery. We will use our established methods as well as novel approaches and unique reagents to test our hypothesis. Experiments will be performed with HIV-infected humanized mouse models and in vitro T cell development and migration assays as proposed in the following specific aims: 1. To investigate the effect of HIV-induced immune activation on S1P and its receptors on entry of CD34+ hematopoietic stem cells into the thymus. 2. To determine whether HIV infection affects egress of nave T lymphocyte subsets from the thymus to the periphery through changes in S1P receptor expression. The present proposal represents a unique collaborative approach to elucidate the role of HIV induced immune activation on T cell development and reconstitution of peripheral naive T cells in HIV-1 infection. Insights into the mechanisms underlying the impairment of precursor entry into and exit of naive T cells out of the thymus will enable the development of immunomodulatory therapeutic interventions to counteract impaired T cell development and regeneration.